Mouse models of human genetic diseases have been critical for confirming that particular mutations cause disease, for obtaining a detailed molecular understanding of the pathophysiology of these diseases, and for developing and testing potential therapies. Technical limitations, however, currently prevent us from generating accurate mouse models of human diseases caused by mutations in our mitochondria! DNA. Although several groups have shown that they can generate mice that contain mitochondrial genomes isolated from mouse tissue culture cells, the technology has not yet been developed that allows us to introduce specific nucleotide changes into mouse mitochondrial genomes. We are proposing to overcome this technical limitation. We have cloned the complete mouse mitochondrial genome stably in E. coli and have shown that we can introduce essentially any desired mutation into these clones. We have also shown that we can introduce these engineered mitochondrial genomes into the mitochondria of yeast cells devoid of their own mtDNA and that these mouse mtDNA genomes are replicated accurately in these cells. We now propose to use these transgenomic yeast mitochondria as vectors for transferring the engineered mtDNA genomes back into the mitochondrial networks of mouse tissue culture cells, which will then be used to generate mice with modified mitochondrial genomes. Our specific aims for this project are to 1) develop and optimize methods for using transgenomic yeast mitochondria as mitochondrial delivery vectors that will efficiently fuse to mouse mitochondrial networks, and to 2) determine how to best screen or select for mouse cells that have been transformed with modified mitochondrial genomes.